Test element with nanofibers

ABSTRACT

The invention concerns test elements, in particular diagnostic test elements, for determining the presence or concentration of biological, medical or biologically or medically effective substances including nucleic acids, proteins, viruses, microorganisms and cells, characterized in that these test elements contain nanofibers.

RELATED APPLICATIONS

This is a continuation application of International ApplicationPCT/EP2005/013066, filed Dec. 6, 2005, which claims priority to DE 102004 058 924.0, filed Dec. 7, 2004, which are hereby incorporated byreference in their entirety.

BACKGROUND

The invention concerns test elements, in particular diagnostic testelements, for determining the presence or concentration of biological,medical or biologically or medically effective substances includingnucleic acids, proteins, viruses, microorganisms and cells,characterized in that these test elements contain nanofibers.

Diagnostic test elements, and in particular, test strips, contain a widevariety of fiber-based materials. Papers or fleeces are especiallynoteworthy. Fleeces in particular are used to separate undesired samplecomponents. As an example, reference is made to blood separation fleecesin glucose tests or in the test strips from, e.g., the Reflotron®system. The fibers that are used in papers or fleeces of the prior artare characterized by diameters between about 5 μm and 200 μm.

Nanofibers have been basically known since about 1930. They are producedby the so-called electrospinning process in which a thin fiber isproduced by applying a high voltage in the range of 10-55 kV to adroplet of a polymer solution or polymer melt (Formhals, A., U.S. Pat.Nos. 1,975,504; 2,160,962; 2,187,306).

Nanofibers have extremely small diameters. Fibers having diameters of10-2000 nm are obtainable depending on the material. In some casesbranched fibers are obtained or fibers are obtained which contain avariable number of polymer beads which can be of different sizes on thefibers. Important influencing variables are known to a person skilled inthe art or may be found in the pertinent literature (e.g., Li and Xia,Adv. Matter. 16 (2004), 1151-1170).

The use of nanofibers in medical products is described in U.S.Publication No. 2003/0171053. This publication concerns a medical devicewhich is covered with a nanofiber layer in order to improvebiocompatibility. Other publications relate to a brain probe which iscoated with nanofibers to improve the biocompatibility and measuringstability (e.g., U.S. Publication No. 2002/0106496 or DiPaolo et al.,Proc. 2^(nd) Joint EMBS/BMES Conf. Houston Tex., USA, 23-23, October2002).

U.S. Publication No. 2003/0217928 discloses a process for theelectrosynthesis of nanostructures which can be used to detect ananalyte also within an electrically conductive array.

WO 02/40242 describes a process for producing products for medical andcell-biological use, e.g., stents by electrospinning nanofibers based oncollagen.

WO 03/026532 describes the use of nanofibers for medical devices, forexample, balloons, catheters, filters and stents.

WO 03/087443 discloses a process for applying nanofibers to an object,for example medical devices such as stents, or devices for thecontrolled release of drugs.

Feng et al. (Angew. Chem. Int. Ed. 42 (2003), 800-802) and Feng et al.(Angew. Chem. Int. Ed. 41 (2002), 1221-1223) describe the production of“superhydrophobic” surfaces made of short nanofibers.

The use of fibers of the usual diameter in test elements for diagnosticapplications has specific disadvantages, especially relating to theseparation of blood cells. The fiber matrices in known test elementstypically have relatively coarse, non-uniform porosity. Blood cells areeither not retained by the large pores or they are retained in theinterior of a fabric layer or fleece. The large pores cause lysis due tothe high capillarity or due to injury or rupture of the membrane of redblood corpuscles on sharp corners and edges, especially in the case ofglass fiber fleeces. Another disadvantage of fleeces or fabrics of theprior art is that these materials become relatively thick and retaincorrespondingly large volumes of liquid in the interstitial space of thefibers. This is problematic in the context of the development trendtowards smaller and smaller sample volumes.

Structures made of hydrophobic conventional fibers can be used as liquidbarriers. Examples of these are known under the trade name Tyvek®.However, a disadvantage of these structures is that an aqueous solutionwhich comes into contact with these fabrics rolls off the surface, whichis why they are not able to penetrate into the pores.

SUMMARY AND DESCRIPTION

The present invention provides test elements for detecting an analytewhich at least partially eliminate the disadvantages of the prior art.More particularly, the invention relates to the use of nanofibers forthe production of test elements, e.g., test strips, arrays or sensors.Nanofibers in the sense of the present invention include electrospunand/or continuous fibers. The fibers preferably have a diameter of10-2000 nm, particularly preferably of 10-1000 nm, and most preferablyof 10-500 nm. Preferred electrospun or continuous nanofibers can bemanufactured in any length. For an application in test elements, thefibers preferably at least have a length of ≧1 mm, particularlypreferably of ≧2 mm. This is in contrast to the short nanofibersdescribed by Feng et al. (2002), supra, and Feng et al. (2003), supra.

The nanofibers in the context of these teachings can be hydrophilicnanofibers, hydrophobic nanofibers and mixtures thereof.

The nanofibers are manufactured from polymers by an electrospinningprocess. Suitable processes are disclosed in the aforementioneddocuments of the prior art. Examples of suitable polymers are organicpolymers including polyolefins such as polyethylene, polypropylene,cycloolefin polymers such as Topas®, polypentene or copolymers thereof,fluorinated or partially fluorinated polymers such as polytetrafluoroethylene or others, polyesters such as polyterephthalate,polyamides such as poly-ε-caprolactam, polyurethanes, polyaromaticcompounds such as poly[p-xylylene] and derivatives thereof, polyvinylalcohols, polyvinylamines, polyethyleneimines, polyalkylene oxides suchas polyethylene oxides or combinations or copolymers thereof.Furthermore, it is also possible to use inorganic nanofibers such asnanofibers based on oxides such as silicates, e.g., glass such assilicate, alkali silicate, quartz or water glass, or nanofibers based onmetal alkoxy condensates or combinations thereof. Combinations oforganic and inorganic nanofibers can also be used.

The nanofibers as components of analytical test elements can be providedin the form of fleeces, fabrics, membranes, layers or combinationsthereof. As mentioned above, such materials can be produced byelectrospinning polymers from solution or from a melt.

Such a nanofiber material can be produced by depositing the fibers in adisordered manner. It is also possible to deposit the fibers in a moreor less ordered manner in order to achieve isotropic or anisotropiceffects. The material properties can be influenced within wide limits bythe selection of the material as well as by the selection of fiberdiameter, fiber density, and spinning parameters.

Such a nanofiber material can be applied to a test element, e.g., a teststrip, by simply spinning the fibers onto the surface. They can also becalendered onto the surface or applied to an adhesive layer such as anacrylate adhesive, a contact adhesive or an adhesive tape. It is alsopossible to partially solubilize the support material by a solvent andto deposit fibers onto the swollen material, just as it is possible toachieve a dissolving effect on the surface of the support using asuitable solvent during the production of the fibers, which then resultsin a permanent bond between the nanofibers and the surface after thesolvent has evaporated. This occurs, for example, more easily when theconditions are selected such that nanofibers with beads are formed. Itis, however, also readily possible to mix these fibers with other fibersfrom another nozzle or, after applying a first layer containing beads,which results in a particularly good bond with the support material, toapply an additional layer comprising fibers of a different design andthickness and/or material.

The test element which contains the nanofibers can, for example, be atest strip, an array or a sensor such as an electrochemical sensor.Nanofibers can be applied to porous or non-porous materials of the testelement.

In one embodiment, the test element contains a test strip whichcomprises at least one porous support material, for example, in the formof a paper, a fleece and/or a membrane, and the nanofibers can beapplied to at least one surface of such a porous support material.

Deposition of the nanofibers onto a conventional paper or fleece or amembrane enables the surface of this support material to be modified insuch a manner that a substantially finer pore size is achieved on thecoated upper side. This allows completely different and substantiallyimproved filter properties to be achieved. Material modified in thismanner can be applied in a known manner to a test strip by gluing orlaminating, etc. On the other hand, the material of the test strip orindividual components thereof can also be composed completely ofnanofibers.

For example, nanofibers can be used in filter elements to separateparticulate components from a sample. In one exemplary embodiment, thefilter element is an element for separating blood cells, preferablyerythrocytes.

In so doing, it is possible to eliminate the tendency for hemolysis byusing nanofibers in blood separation fleeces because the fine fiberssupport the erythrocyte membrane and the erythrocyte membrane is not tomdue to capillary activity. It is also virtually impossible for the fewfine fiber ends of nanofibers to damage the membrane of erythrocytes andthus cause hemolysis. It is possible by using a fleece made ofnanofibers to prepare a very thin fleece, e.g., having a thickness of0.02 μm to 50 μm, preferably 0.05 μm to 5 μm, particularly preferably0.08 μm to 2 μm, with a high filter effectiveness which is then alsoable to process very small volumes of blood and itself has only a verysmall retention. In this application, hydrophilic polymers can be used,such as polyamides, polyurethanes, polyvinyl alcohols, polyvinylamines,polyethyleneimines, polyethylene glycols or copolymers thereof, e.g., ofpolyurethane and polyethylene glycol in order to produce nanofiberstherefrom by electrospinning. Equally preferred are inorganic materialssuch as oxides, preferably glasses such as quartz, silicate, alkalisilicate, water glass, metal alkoxy condensates, or combinationsthereof.

In another exemplary embodiment, nanofibers can be applied to thesurface of a support material in order to modify its properties and inparticular with regard to its ability to be wetted with liquids. Thus, ahydrophobic surface, e.g., a non-porous surface such as the test fieldof an array, can be coated with hydrophilic nanofibers to increase itswettability. A hydrophilic surface in this context preferably has anintrinsic contact angle of <90° with water.

Intrinsic contact angle denotes the contact angle on an ideally smoothsurface which is used as a measure for the surface energy determined bychemical groups without any influence by the surface geometry.

For example, the wetting properties can be dramatically changed bydepositing nanofibers on surfaces. Whereas a drop of water forms acontact angle of about 70-80 degrees on pure poly(methyl methacrylate)PMMA surfaces, a drop of water spreads on a PMMA surface which has beenpartially covered with a thin layer of nanofibers made of polyamide(PA). An amount of 10-500 mg/m², in particular of 50-300 mg/m², e.g.,about 200 mg/m², nanofibers, e.g., made of poly-ε-aminocaprolactamhaving a thickness of 20-2000 nm, e.g., 600 nm has proven to beparticularly advantageous.

On the other hand, non-porous surface such as a test strip housing, inparticular in the region of the sample application zone or an areabetween the test fields of an array, etc., can be coated withhydrophobic nanofibers in order to reduce the wettability of the surfaceand to produce a surface having hydrophobic or super-hydrophobicproperties. In this connection, a hydrophobic surface in the sense ofthe present invention preferably has an intrinsic contact angle of ≧90°with water. A superhydrophobic surface in this context preferably has acontact angle of ≧140°, preferably of ≧150° with water.

A nanofiber coating as a hydrophobic barrier having superhydrophobicproperties is of special interest when developing a hygienic test stripwhere it is absolutely essential to prevent sample liquid such as bloodfrom remaining adhered to the test strip. In order to achieve this, ananofiber structure is manufactured by electrospinning from ahydrophobic base material, e.g., a fluorinated or partially fluorinatedpolymer such as polytetrafluoroethylene (PTFE), a modified soluble PTFEsuch as Teflon® AF, a copolymer of tetrafluoroethylene andhexafluoropropylene (FEP), partially fluorinated polyurethanes,fluorinated polyaromatic compounds or others, or a polyolefin PP,polypentene or others, or polyolefin copolymers. Even a very thin, e.g.,2 μm, layer of such fiber materials or of fibers with beads can be usedto create a super-hydrophobic surface on which a drop of water has acontact angle of ≧140°. A drop of water placed on such a surface alreadyrolls off at a slope of below 20°. A drop of blood which is brought intocontact with the surface of such a layer exhibits no tendency to wetthis layer or adhere to this layer.

One exemplary embodiment is a test element comprising (a) at least onearea covered with hydrophilic nanofibers and (b) at least one areacovered with hydrophobic nanofibers. The areas covered with hydrophilicnanofibers are preferably test fields which are provided for theapplication of sample liquids such as blood in order to improve theirwetting. The areas covered with hydrophobic nanofibers are preferablyarranged in the vicinity of the test fields and/or sample applicationsites in order to prevent an undesired wetting with sample liquid.

In yet another embodiment, a mixture of hydrophilic and hydrophobicnanofibers can be applied to a surface, e.g., to a non-porous surfacefor a more uniform distribution of liquids over the surface. Examples offiber mixtures are Teflon® AF and poly(urethane-g-ethylene oxide).

By applying a thin layer of a mixture of hydrophilic and hydrophobicnanofibers it is possible to modify a surface in such a manner that theapplied drop of liquid and in particular an aqueous drop is uniformlydistributed. Surprisingly, it has been found that when such a dropdries, substances dissolved therein form a substantially more uniformlayer than without the presence of nanofibers. In this manner it ispossible to distribute a test chemistry applied in liquid form in asubstantially more uniform manner than previously, e.g., on an electrodeof an electrochemical sensor. This is also of importance for theproduction of arrays, e.g., that are used in molecular-diagnosticanalyses. In particular, the aspect of self-fluorescence plays a majorrole in these applications. The extremely small amount of material,e.g., 10-500 mg/m² which is necessary for the effect results in only avery low self-fluorescence, which in turn has a favorable effect on thesignal-to-noise ratio when evaluating the arrays.

These teachings also may be applied in a method for the qualitativeand/or quantitative determination of an analyte in a sample in which atest element as described above is used. The method can be animmunochemical method or a method based on nucleic acid hybridization oralso an enzymatic method. Applications include electrochemical and/orphotometric detection methods, e.g., for detecting glucose in blood orother body fluids.

Yet a further application of these teachings is the use of nanofibermaterial, preferably of electrospun and/or continuous nanofibers asdescribed above, as a filter in a test element which is used to detectanalytes. The filter can contain the nanofibers as such and/or they canbe applied to a porous support material as mentioned above. If thefilter contains the nanofibers as such, i.e., in an unsupported form,the material is present as an optionally asymmetric membrane. Asmentioned above, the material of the membrane can contain hydrophilicnanofibers, hydrophobic nanofibers or mixtures thereof.

Advantageously, fleeces or filter materials can be produced for use ontest strips which are substantially thinner and have a much finer andmore uniform pore size and require less material than the materialsaccording to the state of the art. It is also advantageous that thewettability of surfaces can be dramatically improved by nanofibers.Another advantage is that superhydrophobic surfaces can be produced fromhydrophobic polymers on which a drop of water or drop of blood does nothold but rather rolls off at a slight angle. It is also advantageousthat virtually no hemolysis occurs when blood is separated. Nanofibersenable detection reagents to be applied to surfaces in a substantiallymore homogenous manner than was previously the case and theself-fluorescence can be reduced.

While exemplary embodiments incorporating the principles of the presentinvention have been disclosed hereinabove, the present invention is notlimited to the disclosed embodiments. Instead, this application isintended to cover any variations, uses, or adaptations of the inventionusing its general principles. Further, this application is intended tocover such departures from the present disclosure as come within knownor customary practice in the art to which this invention pertains andwhich fall within the limits of the appended claims.

1. A test element for detecting an analyte in a liquid sample,comprising a detection reagent for detecting an analyte, the detectionreagent and nanofibers applied to a porous or non-porous supportmaterial, at least some of the nanofibers containing more than onepolymer bead thereon and having a length of at least 2 mm.
 2. The testelement of claim 1, further comprising continuous nanofibers.
 3. Thetest element of claim 1, wherein the nanofibers are obtained byelectrospinning.
 4. The test element of claim 1, wherein the nanofibershave a diameter of 10 to 2000 nm.
 5. The test element of claim 1,wherein the nanofibers have a diameter of 10 to 1000 nm.
 6. The testelement of claim 1, wherein the nanofibers have a diameter of 10 to 500nm.
 7. The test element of claim 1, wherein the nanofibers comprisehydrophilic nanofibers, hydrophobic nanofibers or mixtures thereof. 8.The test element of claim 7, wherein the hydrophobic nanofibers form asurface of the test element having superhydrophobic properties.
 9. Thetest element of claim 1, wherein the nanofibers are composed of polymersselected from polyolefins, polyaromatic compounds, fluorinated orpartially fluorinated polymers, polyesters, polyamides, polyurethanes,polyvinyl alcohols, polyvinyl amines, polyethyleneimines, polyalkyleneoxides and combinations and copolymers thereof.
 10. The test element ofclaim 1, wherein the nanofibers comprise fleeces, fabrics, membranes,layers or combinations thereof.
 11. The test element of claim 1, whereinthe test element comprises a test strip.
 12. The test element of claim1, wherein the test element comprises a test array.
 13. The test elementof claim 1, wherein the nanofibers are applied to a porous supportmaterial selected from papers, fleeces and membranes.
 14. The testelement of claim 13, wherein the nanofibers are applied to at least onesurface of the porous support material.
 15. The test element of claim13, wherein the nanofibers comprise a filter element for separatingparticulate components from a sample.
 16. The test element of claim 15,wherein the filter element is configured for separating blood cells froma blood sample.
 17. Test element according to claim 1, wherein thenanofibers are applied to the surface of a non-porous support material.18. The test element of claim 1, further comprising a surface coatedwith hydrophilic nanofibers to increase the wettability of the surface.19. The test element of claim 1, wherein the porous or non-poroussupport material comprises a surface coated with hydrophobic nanofibersto reduce the wettability of the surface.
 20. The test element of claim19, wherein the coated surface has superhydrophobic properties.
 21. Thetest element of claim 1, further comprising at least one first areacovered with hydrophilic nanofibers and at least one second area coveredwith hydrophobic nanofibers.
 22. The test element of claim 1, furthercomprising a surface coated with a mixture of hydrophilic andhydrophobic nanofibers, the surface being configured for uniformdistribution of liquids applied thereto.
 23. A test element fordetermining a quantitative or qualitative property of a liquid sample,comprising a detection reagent for detecting an analyte, the detectionreagent, a support material and nanofibers adhered to the supportmaterial, at least some of the nanofibers containing more than onepolymer bead thereon and having a length of at least 2 mm.
 24. The testelement of claim 23, wherein the nanofibers comprise a filter configuredto separate red blood cells from blood in a blood sample.
 25. The testelement of claim 24, wherein the filter comprises a fleece.
 26. The testelement of claim 24, wherein the fleece has a thickness of 0.02 μm to 50μm.
 27. The test element of claim 24, wherein the fleece has a thicknessof 0.08 μm to 2 μm.
 28. The test element of claim 24, wherein thenanofibers are hydrophilic.
 29. The test element of claim 23, whereinthe support material is hydrophobic and the nanofibers are hydrophilic.30. The test element of claim 23, wherein the nanofibers comprise a thinlayer having a mass per unit area of 10 to 50 mg/m².
 31. The testelement of claim 23, wherein the nanofibers comprise an asymmetricmembrane.
 32. The test element of claim 23, wherein the nanofiberscomprise a thin layer having a thickness of 20 to 2000 nm.
 33. The testelement of claim 23, wherein the nanofibers comprise a hydrophobicbarrier.
 34. The test element of claim 23, wherein the nanofiberscomprise a mixture of hydrophilic and hydrophobic nanofibers configuredto uniformly distribute a liquid sample.
 35. The test element of claim23, wherein the nanofibers are disposed on a paper, fleece or membrane.